CN101874119B - 3.4 kb mitochondrial DNA deletion for use in the detection of cancer - Google Patents

Abstract

A method is described wherein prostate or breast cancer is detected by quantifying a 3 4-kilobase deletion in the mitochondrial DNA (mtDNA) of a test sample. The deletion is located between nucleotides 10744-14124 of the mitochondrial genome. An increase in the amount of the deletion relative to the amount of the deletion in non-cancerous prostate and breast tissue is indicative of prostate and breast cancer, respectively.

Description

3.4kb mitochondrial DNA deletion is for detecting cancer

The cross reference of related application

The application is the part continuation application of the PCT application No.PCT/CA2006/000652 that on April 18th, 2006 submits to, require U.S. Provisional Application No.60/672,016 (submission on April 18th, 2005), 60/721, the right of priority of 522 (submissions on September 29th, 2005) and 60/789,872 (submissions on April 7th, 2006).Whole disclosures of these applications are incorporated to herein all by reference.

Technical field

The present invention relates to Mitochondrial Genome Overview field.Especially, it relates to 3.4kb disappearance and the application as cancer indicator thereof in Mitochondrial Genome Overview.

Background technology

As the Mitochondrial DNA (mtDNA) of diagnostic tool

MtDNA Nucleotide Sequence Dynamics is important diagnostic tool.Sudden change in mtDNA is usually the preliminary indicator that disease occurs, often suddenly change relevant to nucleus, and be used as biomarker, its particularly: disease, such as but not limited to smoking and be exposed to the tissue injury and cancer (Lee etc., 1998 that second hand smoking causes; Wei, 1998); Life-span, based on about 20 years old start and increase thereafter Mitochondrial Genome Overview sudden change accumulation (von Wurmb, 1998); By suddenly change or be exposed to carcinogens, metastatic disease (Birch-Machin, 2000) that mutagenic compound, ultraviolet radiation cause; Osteoarthritis; Cardiovascular diseases, alzheimer's disease, Parkinson's disease (Shoffner etc., 1993; Sherratt etc., 1997; Zhang etc., 1998); Age related hearing disability (Seidman etc., 1997); Optic nerve degeneration and irregular pulse (Brown etc., 1997; Wallace etc., 1988); Chronic progressive external exophthalmic ophthalmoplegia (external exophthalmoplegia) (Taniike etc., 1992); Atherosclerosis (Bogliolo etc., 1999); Papillary thyroid carcinoma and thyroid tumor (Yeh etc., 2000); Etc. (such as Naviaux, 1997; Chinnery and Turnbull, 1999).

The sudden change of Mitochondrial Genome Overview specific site can with some disease-related.Such as, the sudden change of the 4216th, 4217 and 4917 and leber hereditary optic neuropathy (Leber ' s Hereditary Optic Neuropathy, LHON) relevant (Mitochondrial Research Society; Huoponen (2001); MitoMap).The 15452nd sudden change relevant to ubiquinone cytochrome C-reductase (Complex II I) defect (Valnot etc., 1999) is found in 5/5 patient.

Particularly, these sudden changes or change comprise point mutation (conversion, transversion), disappearance (base is to several thousand bases), inversion, copy (base is to several thousand bases), restructuring and insert (base is to several thousand bases).In addition, find that specific base pair changes, lack or it combines and the early onset thereof of prostate cancer, skin carcinoma and lung cancer and aging (such as Polyak etc., 1998), early ageing, to be exposed to carcinogens (Lee etc., 1998) etc. relevant.

Prostate cancer

Prostate cancer is often by the solid tumor diagnosed out, and it most possibly derives from prostatic epithelium (Huang etc., 1999).In 1997, having screened prostate specific antigen (Prostate specific antigen, PSA) to nearly 10,000,000 American male, there is prompting and suffers from prostate cancer (Woodwell, 1999) in it.In fact, this shows that the digital rectal examination (digital rectalexam, DRE) by preliminary can filter out the male sex of more high number.Same year, 31,000,000 male sex have carried out DRE (Woodwell, 1999).In addition, the cases for prostate cancer number that the U.S. newly diagnoses out is every year estimated as 179,000 (Landis etc., 1999).It is the second cancer the most often diagnosed out and cause the second main cause of cancer mortality in the Canadian male sex.In 1997, in the cancer that prostate cancer is newly diagnosed out the Canadian male sex, account for 19,800 examples (28%) (Canadian National Cancer Institute).According to estimates, in the male sex of all more than 49 years old, 30% to 40% has some cancerous prostate cells, but prostate cancer (the SpringNet-CE Connection only having 20% to 25% to have a clinical remarkable form in these male sex, internet, www.springnet.com/ce/j803a.htm).Prostate cancer shows as Various Tissues scholarship and moral conduct, comprises internal factor and externalities, and namely socioeconomics, diet, geographical environment, hormone imbalances, family history and gene form (Konishi etc., 1997; Hayward etc., 1998).Although it is relevant to prostate cancer previously to have found that some mtDNA changes, still need other mark for detecting prostate cancer.

3.4 kb mtDNA lack and prostate cancer detects.

PCT application (the publication number WO/06/111029 of inspection phase is still in the applicant, its full content is incorporated to herein by reference) in, the disappearance of the 3379bp fragment of mtDNA is identified by carrying out full Mitochondrial Genome Overview amplification to prostata tissue.After measured, this 3379bp lacks (being called " 3.4kb disappearance ") between 10744 ~ 14124 Nucleotide of Mitochondrial Genome Overview.Confirm to utilize when test organization sample and diagnosing prostate cancer is come to the detection of this disappearance.

It is all or part of that described 3.4kb disappearance eliminates in following gene from mtDNA genome: (i) nadh dehydrogenase subunit 4L, (ii) nadh dehydrogenase subunit 4, (iii) nadh dehydrogenase subunit 5, (iv) tRNA Histidine, (v) tRNA Serine 2, and (vi) tRNA leucine 2.

Mammary cancer

Mammary cancer is the cancer of mammary tissue, and it occupy the 5th of cancer mortality common cause.In 2005, mammary cancer caused 502 in worldwide, 000 people's death (accounting for 7% of cancer mortality, 1% of nearly all death) (World Health Organization cancerous condition specification sheets No.297).In global women, mammary cancer is modal cancer and modal cancer mortality reason (World Health Organization cancerous condition specification sheets No.297).Although it is relevant to mammary cancer previously to have found that some mtDNA changes, such as Parrella etc. (Cancer Research:61,2001), still need other mark for detecting mammary cancer.

Summary of the invention

In one embodiment, the invention provides the method detecting cancer in individuality, it comprises:

A) biological sample is obtained from described individuality;

B) from this sample extraction Mitochondrial DNA (mtDNA);

C) the quantitative amount of this mtDNA in sample, has disappearance in its nucleotide sequence between mtDNA genome 10744 ~ 14124 residues;

D) amount and at least one known reference point in sample with the mtDNA of described disappearance is compared.

In one embodiment, the invention provides the method detecting cancer in individuality, it comprises:

A) biological sample is obtained from described individuality;

B) from this sample extraction Mitochondrial DNA (mtDNA);

C) the quantitative amount of this mtDNA in sample, has disappearance in its nucleotide sequence between mtDNA genome 10744 ~ 14124 residues;

D) amount in sample with the mtDNA of described disappearance is compared with reference to the amount lacked described in sample with the mtDNA from known non-cancerous tissue or body fluid;

Wherein, with compared with sample, the raising instruction cancer of the amount lacked described in biological sample.

In one embodiment, the invention provides the method detecting cancer in individuality, it comprises:

A) biological sample is obtained from described individuality;

B) from this sample extraction Mitochondrial DNA (mtDNA);

C) the quantitative amount of this mtDNA in sample, has disappearance in its nucleotide sequence between mtDNA genome 10744 ~ 14124 residues;

D) amount in sample with the mtDNA of described disappearance is compared with reference to the amount lacked described in sample with the mtDNA from known cancerous tissue or body fluid;

Wherein, the level lacked described in biological sample with reference to similar in sample, indicate cancer.

In one embodiment, the invention provides the method that monitoring individual cancer occurs, it comprises:

A) biological sample is obtained;

B) from this sample extraction mtDNA;

C) the quantitative amount of this mtDNA in sample, has disappearance in its nucleotide sequence between mtDNA genome 10744 ~ 14124 residues;

D) within for some time repeating step a) to c);

E) wherein within the described time period, the raising of described disappearance level indicates cancer.

In one embodiment, the invention provides the method detecting cancer in individuality, it comprises:

A) biological sample is obtained from described individuality;

B) from this sample extraction Mitochondrial DNA (mtDNA);

C) the quantitative amount of this mtDNA in sample, it has the sequence corresponding to sequence SEQ ID NO:1;

D) amount and the known reference point of at least one that correspond to the mtDNA of SEQ ID NO:1 in sample are compared.

Accompanying drawing explanation

Embodiment of the present invention are described by means of only citing and with reference to the mode of accompanying drawing, wherein:

Fig. 1 signal can be used for design and the sequence of the primer detecting described 3.4kb disappearance.

Fig. 2 to be presented in 3.4kb research the comparing of cycle threshold between pernicious participation object with the optimum participation object of symptom.

Fig. 3 shows cycle threshold involved in embodiment 1.

Fig. 4 shows ROC curve, the specificity of its signal one embodiment of the invention and sensitivity.

Fig. 5 shows ROC curve, the specificity of its signal another embodiment of the present invention and sensitivity.

Fig. 6 display relates to the real-time PCR data of the 3.4kb mtDNA disappearance level relevant to mammary cancer.

Fig. 7 shows ROC curve, the specificity of its signal another embodiment of the present invention and sensitivity.

Detailed Description Of The Invention

" cycle threshold " used herein (C _t) be the point using the target of PCR in real time to increase when rising to more than background, as passed through indicated in signal (such as fluorescent signal).C _twith the amount of sequence studied be negative correlation.

" sensitivity " defined herein refers to the mark (True Positive Rate) of the true-positive results using the inventive method to obtain.

" specificity " defined herein refers to the mark (false positive rate) of the false positive results using the inventive method to obtain.

In one embodiment of the invention, the method being carried out detection and diagnosis cancer by detection and quantitative above-mentioned 3.4kbmtDNA disappearance is provided.Such as, the present invention can be used for detecting the front tumorigenesis (pre-neoplasia) of prostate cancer and mammary cancer, tumorigenesis and to possible malignant prostate cancer and breast cancer development.On the one hand, the present invention includes detect and quantitative described 3.4kb mtDNA lack (SEQ ID NO:1) for detecting, diagnosis and/or monitor cancer.In the method, mtDNA extracts biological sample (such as body tissue or body fluid (as urine, massage of prostate liquid (massagefluid))).Then, the level (that is, measuring) that the mtDNA that test is extracted lacks with 3.4kb in working sample.In the test that the present inventor carries out, find that disappearance level described in the sample obtained from the object suffering from cancer improves compared with the sample that the object of never cancer stricken obtains.Based on the following information that provides and data, the present inventor draws such conclusion: in mtDNA, 3.4kb lacks the raising instruction cancer of level.

As disclosed in PCT WO/06/111029, described 3.4kb disappearance approximately covers genomic 10744 ~ 14124 Nucleotide of mtDNA.Described mtDNA genome shows for SEQ ID NO:8 (Genbank accession number AC_000021).As Examples below provide, the present inventor has determined that this disappearance is also relevant to cancer (particularly prostate cancer and mammary cancer).Therefore, this disappearance provides accurate biomarker, and the valuable instrument for detecting, diagnosing or monitor the cancer at least these tissues thus.

Described disappearance causes generation two kinds to lack monomer, and wherein a kind of size is 3.4kb (little sublimon), and alternative size is about 12.6kb (large sublimon).By identify little sublimon existence or by determine described 3.4kb sequence from large sublimon disappearance detect the generation of disappearance.

As discussed above, described disappearance is about 3379bp, and it comprises the gene of coding nadh dehydrogenase subunit 4L, nadh dehydrogenase subunit 4, nadh dehydrogenase subunit 5, tRNA Histidine, tRNA Serine 2 and tRNA leucine 2.

In one embodiment, within for some time, (such as several years) obtains the sample of such as prostata tissue, massage of prostate liquid, urine, mammary tissue from individuality and tests it, to monitor generation or the development of cancer.The raising in time of described 3.4kb disappearance level can indicate beginning or the development of cancer.

The age related progressive that described 3.4kb mtDNA lacks can make individuality easily suffer from such as prostate cancer or mammary cancer, these two kinds of cancers ubiquity in middle-aging male and middle and aged women respectively.According to an aspect of the present invention, provide a kind of method, the amount wherein lacked in time by 3.4kb described in monitoring machine body tissue (such as mammary tissue) or body fluid (as massage of prostate liquid or urine) carrys out customary examination cancer.

System and method of the present invention can be used for the cancer before detecting early-stage cancer and the abnormal appearance of any histology.Such as, system and method for the present invention can be used for detecting the front tumorigenesis in mammary tissue.

The following primer sequence of preferred use detects described 3.4kb and lacks:

3.4 forwards (in conjunction with genomic 10729 ~ 10743/14125 ~ 14139 bit bases of mtDNA) 5 '-TAGACTACGTACATACTAACCCTACTCCTA-3 ' (SEQ ID NO:2);

3.4 reverse (in conjunction with genomic 14361 ~ 14379 bit bases of mtDNA) 5 '-GAGGTAGGATTGGTGCTGT-3 ' (SEQ ID NO:3).

In one embodiment of the invention, using pair for amplification primer to increase indicates described 3.4kb to lack the target region existed.In this embodiment, amplimer to one of with the mtDNA montage region overlapping (that is, the spliceosome of mtDNA genome 10744th ~ 14124) occurred after described 3.4kb sequence deletion.Therefore, the extension of described overlapping primers only just may be there is when described 3.4kb excalation.

In another embodiment of the present invention, pair for amplification primer is used to increase the target region relevant to described 3.4kb deletion sequence.Described 3.4kb deletion sequence, once disappearance, just can form ring-type mtDNA molecule again.In this embodiment, amplimer to one of overlap with the site that reconnects of described 3.4kb sequence end.Therefore, the amount instruction cancer of the 3.4kb molecule of increase is detected in the sample to which.The following primer pair of preferred use detects the 3.4kb nucleic acid of described disappearance:

Forward 14,115,/10,755 5 '-CCCACTCATCACCTAAACCTAC-3 ' (SEQ ID NO:9)

Reverse 10980R 5 '-GGTAGGAGTCAGGTAGTTAG-3 ' (SEQ ID NO:10).

In one aspect of the invention, provide the test kit for diagnosing cancer such as prostate cancer or mammary cancer, it comprises the instrument/device for extracting mtDNA, the primer with nucleotide sequence shown in SEQ ID NO:2 and 3 or SEQ ID NO:9 and 10, reagent and explanation.

Another aspect provides the method for the biopsy test of cancer existence in checking or negative biopsy samples (such as prostate cancer or mammary cancer), it comprises: obtain non-cancerous tissue from biopsy samples; Detect also 3.4kb mtDNA described in quantitative non-diseased tissue to lack.

In one embodiment, the invention provides the method whether suffering from prostate cancer or mammary cancer from humoral sample examination individuality, it comprises: obtain humoral sample, detects and the level of 3.4kbmtDNA disappearance described in quantitative body fluid.

Although as described in Examples below, real time quantitative PCR method represents and detects described 3.4kb disappearance and whether exists and the preferred means quantitative to it, but also can use and well known to a person skilled in the art other method.Such as, the Bioplex of Bio-Rad can be used ^tMsystem and suspension array technology are carried out quantitatively described disappearance.Generally speaking, described method needs to use any currently known methods to carry out amplification with quantitative to sequence.

Embodiment provided below illustrates that this disappearance not only can be used for detecting the prostate cancer in prostata tissue, also can be used for the existence detecting cancer in other biological sample (such as massage of prostate liquid, urine and mammary tissue).Based on the discovery in these embodiments, described 3.4kb mtDNA disappearance can be used as the biomarker of cancer.

The multiple embodiments provided illustrate from suffering from the amount between the sample that obtains cancer and not cancered object with the mtDNA that described 3.4kb lacks and there are differences.Find in the sample obtained from the object suffering from cancer, the amount of described 3.4kb disappearance is higher.Above-mentioned conclusion is drawn by the amount of 3.4kb disappearance described in test sample being compared with the amount from known cancer cells and/or known non-cancerous cells.

Embodiment 1: the 3.4kb disappearance in prostata tissue mtDNA

By identifying the disappearance of about 3.4 kilobase (kb) to the plastosome whole genome amplification of fresh food frozen prostata tissue.Use the size of this disappearance of linear regression estimation between 3000 base pairs (bp) to 3500bp.Use Mitomap ^tM(Brandon, M.C., Lott, M.T., Nguyen, K.C., Spolim, S., Navathe, S.B., Baldi, P. & Wallace, D.C., MITOMAP:ahuman mitochondrial genome database--2004 update.Nucleic Acids Research 33 (Database Issue): D611-613,2005; www.mitomap.org) identify two kinds of possible candidate's disappearances, i.e. the 3397bp disappearance of 9574 ~ 12972 and the 3379bp disappearance of 10744 ~ 14124.In order to determine which kind of relevant with prostate cancer (if any) in two kinds of disappearances, crossing over the forward primer of this deletion junction for each design in two kinds of candidate's disappearances, guaranteeing the repeat region of this primer extension by described disappearance flank.Fig. 1 is the schematic diagram of the described design of primers of display and sequence (i.e. SEQ ID NO:2).Obtain the positive amplification result of the amplicon corresponding to 10744 ~ 14124 3379bp disappearance (being called " 3.4kb disappearance ").

As mentioned above, it is all or part of that described 3.4kb disappearance eliminates in following gene: (i) nadh dehydrogenase subunit 4L, (ii) nadh dehydrogenase subunit 4, (iii) nadh dehydrogenase subunit 5, (iv) tRNA Histidine, (v) tRNA Serine 2 and (vi) tRNA leucine 2.

The existence that described 3.4kb lacks is detected in 91% of 33 fresh food frozen prostate samples.Use the tissue that specific deficiency primer test formalin is fixing, to put forward high n-value.

Researchist of the present invention is to from being carried out 32 tissue samples of Microscopic excision by detection wind lidar and checking order from the intact mitochondria genome of 12 needle puncture biopsies of histology normal prostatic.The tissue slice of file from each these sample is used for following research.1 ~ 2 serial section is taken out from each sample.DNA is extracted from the entirety of each sample microsection.Therefore, each sample is made up of the mixture of body of gland prostata tissue and matrix prostata tissue.Use Qiagen ' s QIAamp ^tMthe mini test kit of DNA (article No. 51304) carries out this extraction.After extraction, use Nano-Drop ^tMspectrophotometer carries out quantitatively sample, and concentration is normalized into 2ng/ μ l subsequently.20ng is used to input DNA and iQ ^tMsYBR Green Supermix ^tMincrease test kit (Bio-Rad Laboratories Inc.) each sample.Utilize 2 double-colored real-time PCR systems (MJ Research) react.

As shown in Figure 2, the cycle threshold between malignant prostate sample and symptom benign prostate sample and by extend measure disappearance amount between observe obvious difference.Malignant samples unanimously shows the cycle threshold early than benign sample.

Embodiment 2: to blind research---the comparison of cycle threshold of 3.4kb disappearance

Select other 21 Prostate tissue specimens, wherein 10 is optimum, and 11 is pernicious.Needle puncture biopsy is carried out to determine pathological state by the pathologist with qualification.Described sample is blind, thus investigator of the present invention does not know their pathological state when carrying out this test.By checking cycle threshold, investigator of the present invention correctly can dope the pathological state in 81% case.For 4 error predictions, wherein 2 be malignant samples is defined as optimum, 2 be benign sample is defined as pernicious.Doctor require to obtain in latter event 2 individual follow up a case by regular visits to clinical information, thus determine whether they are diagnosed out prostate cancer after the needle puncture biopsy result of this research use.Wherein obtain benign sample at first but be predicted as a pernicious individuality by this research obtaining malignant samples subsequently.As a result, a false positive becomes true positives.Therefore, in this research detect in case 86% pathological state prediction be correct.This studies final positive predictive value (PPV, wherein PPV=true positives/(true positives+false positive)) is 91%, and negative predictive value (NPV, wherein NPV=true negative/(true negative+false negative)) is 80%.

Embodiment 3:3.4kb lacks research---method (n=76)

In this research, 3.4kb disappearance is detected to 76 Prostate tissue specimens.All tissue samples are all fixed with formalin, and wherein 25 is pernicious, and 12 is normal, and 39 histology displays have benign prostate conditions.In latter one group, over half have hyperplasia.All samples are takes from the needle puncture biopsy that investigator organizes file.

Prostate samples

The Prep-Strips (article No. LCM0207) from Arcturus Bioscience Inc. is used to carry out tapelift on each slide glass.This makes it possible to any particulate matter or the tissue that do not adhere to be removed from slide glass before DNA extraction.Make to organize and be still attached on slide glass, by PBS (phosphate buffered saline(PBS)) washed to remove fixing agent as much as possible.The aseptic operation blade of individual packaging is used the needle puncture biopsy section of 1 ~ 2 on slide glass to be blown in sterile eppendorf tubes.Then, use according to the specification sheets of manufacturers dNA is separated and purifying by the mini test kit of DNA (Qiagen, article No. 51304).Contrast with slide glass extract parallel processing negative extraction, as quality control inspection point.By spectrophotometer (Nano-Drop ^tMnD-1000) measure STb gene concentration and the purity ratio of each sample, and the dilution preparing 2ng/ μ l is for quantitative polyase chain reaction (qPCR).

Primer (oligonucleotide)

By the Oligonucleolide primers of Invitrogen (California, USA) chemosynthesis purifying.The expection size of the PCR primer of primer sequence and amplification is listed in table 1.In addition, positive control (DNA in the known source with sudden change mtDNA) is comprised to the pcr analysis of mtDNA disappearance.Each combination of primers except TNF (tumour necrosis factor) is checked with without mitochondrial rho 0 clone, to confirm the coamplification that there is not pseudogene.

Table 1 amplimer.

Real-time polymerase chain reaction

Three independently PCR are carried out to each sample.The cumulative volume of each reaction is 25 μ l, comprises template DNA, pair of primers (12s or 3.4 disappearance or TNF), iQ ^tMsYBR Green Supermix ^tMtest kit (article No. 170-8882, Bio-Rad Laboratories Inc.) and distillation deionized water (ddH ₂o).TNF (tumour necrosis factor) comprises single-copy nuclear gene primer, and 12s comprises total Mitochondrial Genome Overview primer.Volume and the concentration of template DNA, primer and reaction buffer are listed in the table below.

Table 2 qPCR component.

The loop parameter of each amplicon lists in table 3.

Table 3 loop parameter.

Use is equipped with Intuitive Opticon Monitor ^tMthe DNA Engine of software 2 continuous fluorescence detection systems (MJ Research Inc.) carry out thermal cycling, in real time detection and response analysis.It is quantitative that use calibration curve method carries out DNA.Serial dilution (10 is carried out to three kinds of purified templates that PCR produces ⁶, 10 ⁵, 10 ⁴, 10 ³, 10 ², 10 ¹), a kind of product corresponds to 3.4 disappearances, and a kind of corresponding to 12s primer, one corresponds to TNF.Thereby produce three different typical curves, it shows the amount of the copy number of total mtDNA (12s amplicon-plastosome full-length genome primer), the amount with the mtDNA of 3.4kb disappearance or total nucleus DNA (TNF-single-copy nuclear gene primer).By the C of comparative sample _tthe C of value and standard substance _tvalue, the C of described sample _tthen value can convert DNA copy number to.If 3.4 disappearances do not detected in 37 circulations, then think that this disappearance does not exist or exists with low-level.

Based on exist in described normalization method sample 3.4kb disappearance amount determine pernicious, as described in circulation thresholding position indicated by.This position can be absolute location (as is greater than 25 circulations but is less than 35 circulations), or more may total Mitochondrial DNA existence indicated by 12s amplicon and 3.4kb lack between ratio.This can be expressed as the percentage ratio of total Mitochondrial DNA.The cell count representated by TNF amplicon can be incorporated to, with the differentiation between refinement benign tissue and malignant tissue.

In order to these samples of automated analysis, employ bioinformatics tools.These analyze the cycle threshold C that three variablees related to are tumour necrosis factor (TNF) respectively _t, the total kind of plastosome containing specific primer site and there is the plastosome of object disappearance.

Cluster analysis

Due to the similarity and among a small circle of data, cluster is not normalized, and does not use logarithmic function.

The true movement of Fig. 3 display data and trend.X-axis is patient's number, and y-axis is the cycle threshold that PCR in real time obtains.

Should emphatically point out, cycle threshold is higher, represents that the amount that disappearance exists is lower.

The general trend be shown in Fig. 3 is the difference/ratio based on disappearance, total and TNF variable.This disappearance is low extremely not existing in optimum/normal specimens (right side), in abnormal benign and malignant samples, then raise (to the left).Abnormal benign and malignant samples are distinguished from each other based on the cycle threshold ratio between disappearance and TNF.

Formula is instructed to know (Supervised Learning)

Formula is instructed to know system based on attempting to predict known sample result.Use half logarithmic data training, and second half is for testing algorithm.Instruct formula to know to be predicted and compare with objective result, and " know " from mistake.But if this predicts the outcome higher or lower than the legitimate reading of data, this mistake can be passed back by system and correspondingly adjust weight.

Data set: 5% to 35%-is optimum

35% to 65%-hyperplasia

65% to 95%-is pernicious

Artificial neural network (ANN) algorithm (schematically as follows):

The half of data set is for training ANN

Second half is for more accurate degree.Tolerance range=to the data set of prediction and the data set of acquisition compare → 86.6%

End user's artificial neural networks (ANN) instructs formula to know to missing data

Three classifications:

Optimum

Hyperplasia

Pernicious

Based on PCR in real time cycle threshold C _tthree variablees are used to every kind:

Tumour necrosis factor (TNF)-nucleus copy contrast

Total plastosome-plastosome copy contrast

The plastosome of disappearance-miss status.

Result:

The half of data set is for training ANN, and all the other half are used for more accurate degree.

Tolerance range=86.6% of three kinds of classification

Positive predictive value (PPV);

Optimum to pernicious=88.2%

Negative predictive value (NPV)

Optimum to pernicious=76.5%

Embodiment 4: the 3.4kb disappearance in the mtDNA relevant to mammary cancer

Test from the pernicious existence with above-mentioned 3.4kb disappearance in 18 samples of benign breast tissue, wherein 9 is pernicious, and 9 is optimum.Conventional histopathological analysis is used to be divided into by sample pernicious or optimum.

Use according to the explanation of manufacturers the mini test kit of DNA (Qiagen, article No. 51304) is separated and purify DNA from sample.

By the oligonucleotide of Invitrogen (California, USA) chemosynthesis purifying.List in table 1 above primer sequence and the expection size of the PCR primer that increases.

Real-time polymerase chain reaction

Three independently PCR are carried out to each sample.The cumulative volume of each reaction is 25 μ l, comprises template DNA, pair of primers (12s or 3.4 disappearance or TNF), iQ ^tMsYBR GreenSupermix test kit (article No. 170-8882, Bio-Rad Laboratories Inc.) and distillation deionized water (ddH ₂o).TNF (tumour necrosis factor) comprises single-copy nuclear gene primer, and 12s comprises total Mitochondrial Genome Overview primer.Volume and the concentration of template DNA, primer and reaction buffer are listed in the table below.

Table 4 qPCR component.

The loop parameter of each amplicon is listed in table 5.

Table 5 loop parameter.

Use is equipped with Intuitive Opticon Monitor ^tMthe DNA Engine of software 2 continuous fluorescence detection systems (MJ Research Inc.) carry out thermal cycling, in real time detection and response analysis.It is quantitative that use calibration curve method carries out DNA.Serial dilution (10 is carried out to three kinds of purified templates that PCR produces ⁶, 10 ⁵, 10 ⁴, 10 ³, 10 ², 10 ¹), a kind of product corresponds to 3.4 disappearances, and a kind of corresponding to 12s primer, one corresponds to TNF.Thereby produce three different typical curves, its show total mtDNA (12s amplicon-plastosome full-length genome primer), 3.4 disappearances or total nucleus DNA (TNF-single-copy nuclear gene primer) copy number.By the C of comparative sample _tthe C of value and standard substance _tvalue, the C of described sample _tthen value can convert DNA copy number to.

Based on exist in described normalization method sample 3.4kb disappearance amount determine pernicious, as described in circulation thresholding position indicated by.This position can be absolute location (as is greater than 25 circulations but is less than 30 circulations), or more may total Mitochondrial DNA existence indicated by 12s amplicon and 3.4kb lack between ratio.This can be expressed as the percentage ratio of total Mitochondrial DNA.

In order to these samples of automated analysis, employ bioinformatics tools.These analyze the cycle threshold C that three variablees related to are tumour necrosis factor (TNF) respectively _t, the total kind of plastosome containing specific primer site and there is the plastosome of object disappearance.

Table 6 and Fig. 7 show the average C from malignant tissue and benign tissue _tthe difference of score value.The average C of healthy tissues _tvalue is 30.5889, and the average C of malignant tissue _tbe 27.8533, show that the amount of the mtDNA in malignant breast tissue with described 3.4kb disappearance and normal galactophore tissue there are differences thus.

Table 6 C _tthe mean value of score value

Group statistical study

Fig. 8 is ROC curve, shows that described 3.4kb mtDNA lacks as the specificity of markers for breast cancer during detection mammary tissue and sensitivity.Use C _tcutoff value 29.1900 obtains these results.At this C _tunder, the sensitivity of described mark is 77.8%, and specificity is 77.8%.

Table 7 shows the calculating of the area under curve of the present embodiment.As the tolerance to this test accuracy.

Table 7 shows the result of area under curve

Area under curve

Test result variable: 3.4 disappearances

A. nonparametric hypothesis

B. null hypothesis: true area=0.5

Hereafter table 8 shows C _tthe determination of cutoff value 29.1900.The result display C listed in table 8 _tcutoff value 29.1900 each provides sensitivity and the specificity of 78% and 78%.

Table 8:C _tthe determination of cutoff value.

The coordinate of curve

Test result variable: 3.4 disappearances

A. minimum cutoff value is that the minimum value of observed test value subtracts 1, and maximum cutoff value is that the maximum value of observed test value adds 1.Other cutoff values all are the mean value of two set up Continuous Observation test values.

Embodiment 5: with the massage of prostate liquid phase ratio of individuality without prostate cancer tissue evidence, suffers from the 3.4kb disappearance in the massage of prostate liquid of the individuality of prostate cancer

Collect 40 parts of massage of prostate liquid samples by urologist from patient, described patient is diagnosed as prostate cancer subsequently or after prostate gland needle puncture biopsy step, is not shown the Histological Evidence of prostate cancer.By sample deposition in IsoCode Card ^tMon (Schleicher & Shuell), dry, then extract according to the scheme of manufacturers.Use NanoDrop ^tMnD-1000 spectrophotometer carries out quantitatively, DNA concentration being normalized to 2ng/ μ l to all DNA extraction things.Then according to following parameters, each sample is increased:

In 25 μ l reaction systems,

1×iQ SYBR Green Supermix ^TM(Bio-Rad P/N 170-8880)

150nmol forward primer

(5’-TAGACTACGTACATACTAACCCTACTCCTA-3’)(SEQ ID NO：2)

150nmol reverse primer

(5’-GAGGTAGGATTGGTGCTGT-3’)(SEQ ID NO：3)

20ng template DNA.

According to following scheme, utilize Opticon ^tM2 DNA Engine (Bio-Rad Canada) circulate to reaction system:

1.95 DEG C 3 minutes

2.95 DEG C 30 seconds

3.66 DEG C 30 seconds

4.72 DEG C 30 seconds

5. read plate

6. repeating step 2-5,44 times

7.72 DEG C 10 minutes

8. melting curve is from 50 DEG C to 105 DEG C, every 1 DEG C of reading, keeps 3 seconds

9. keep at 10 DEG C.

Table 9 shows the average C of massage of prostate liquid test _tthe result of value

Group statistical study

Table 9 and table 10 show the average C of benign sample and the acquisition of malignant samples group _tthe significant difference (p=0.005) of value.

Table 10 show sample C _tthe result (p=0.005) that value difference is different.

Independent sample is tested

Fig. 5 is recipient's performance characteristic (Receiver Operating Characteristic, ROC) curve, and it shows that 3.4kb mtDNA lacks as the specificity of prostate cancer marker during detection massage of prostate liquid and sensitivity.Use C _tcutoff value 37.3683 obtains these results.At this C _tunder, the sensitivity of described mark is 87%, and specificity is 64%.

The tolerance range of this test depends on how component one-tenth to be measured is suffered from prostate cancer and not suffered from the group of prostate cancer by this test.Measuring accuracy is carried out by ROC area under a curve.Table 11 shows the calculating of the area under curve of the present embodiment.

Table 11 shows the result of ROC area under curve

Area under curve

Test result variable: 3.4 disappearances

A. nonparametric hypothesis

B. null hypothesis: true area=0.5

The determination of table 12 specificity and sensitivity

The coordinate of curve

Test result variable: 3.4 disappearances

Minimum cutoff value is that the minimum value of observed test value subtracts 1, and maximum cutoff value is that the maximum value of observed test value adds 1.Other cutoff values all are the mean value of two set up Continuous Observation test values.

Show C in table 12 above _tthe determination of cutoff value 37.3683.The result listed in table 12 shows C _tcutoff value 37.3683 provides the highest sensitivity and specificity.

Embodiment 6: with from do not have prostate cancer tissue evidence individuality urine compared with, suffer from the urine of the individuality of prostate cancer 3.4kb disappearance.

A urine sample collection device from 5 diagnosis suffer from the patient of prostate cancer and 5 receive needle puncture biopsy but the individuality that prostate gland cancerates cannot be detected.These samples are collected after digital rectal examination (DRE), are beneficial to the collection of prostatic cell.

After obtaining sample, take out 5ml equal portions, then with 14, the centrifugal 2ml of 000 × g is to form precipitation.Take out and supernatant discarded.Throw out is resuspended by 200 μ l phosphate buffered saline(PBS).According to the explanation of manufacturers, use QiaAMP ^tMresuspended throw out and full urine samples are carried out DNA extraction by the mini test kit of DNA (Qiagen P/N 51304).Then, NanoDrop is used ^tMnD-1000 spectrophotometer carries out quantitatively gained DNA extraction thing, and is normalized to the concentration of 0.1ng/ μ l.

Lack Auele Specific Primer according to hereafter using 3.4kb and utilize quantitative real-time PCR analysis sample:

In 25 μ l reaction systems,

1×iQ SYBR Green Supermix ^TM(Bio-Rad P/N 170-8880)

100nmol forward primer

(5’-TAGACTACGTACATACTAACCCTACTCCTA-3’)(SEQ ID NO：2)

100nmol reverse primer (5 '-GAGGTAGGATTGGTGCTGT-3 ') (SEQ ID NO:3)

1ng template DNA.

According to following scheme, utilize Opticon ^tMreaction system circulates by 2DNA Engine (Bio-Rad Canada):

1.95 DEG C 3 minutes

2.95 DEG C 30 seconds

3.69 DEG C 30 seconds

4.72 DEG C 30 seconds

5. read plate

6. repeating step 2-5,44 times

7.72 DEG C 10 minutes

8. melting curve is from 50 DEG C to 105 DEG C, and every 1 DEG C of reading, keeps 3 seconds

9. keep at 10 DEG C

Table 13 C _tthe mean value of score value

Group statistical study

Table 13 and table 14 show the average C that benign sample and malignant samples group obtain _tsignificant difference (p=0.005) between value.

Table 14 show sample C _tthe result (p=0.005) that value difference is different.

Independent sample is tested

Fig. 6 is recipient's performance characteristic (ROC) curve, and it shows that 3.4kb mtDNA lacks specificity and the sensitivity of prostate cancer marker when urinating as test.Use C _tcutoff value 31.575 obtains these results.At this C _tunder, the sensitivity of described mark is 80%, and specificity is 100%.

Table 15 shows C _tthe determination of cutoff value 31.575.The result display C listed in table 15 _tcutoff value 31.575 provides the highest sensitivity and specificity.

Table 15:C _tthe determination of cutoff value.

The coordinate of curve

Test result variable: CTf

A. minimum cutoff value is that the minimum value of observed test value subtracts 1, and maximum cutoff value is that the maximum value of observed test value adds 1.Other cutoff values all are the mean value of two set up Continuous Observation test values.

Embodiment 7: detect the 3.4kb of the cyclisation again deletion sequence in the pernicious and benign tissue of prostate gland

In this embodiment, the indicator of amount as prostate cancer of cyclisation 3.4kb disappearance mtDNA molecule again in sample is tested.As mentioned above, described 3.4kb sequence again can form the mtDNA molecule of cyclisation after disappearance.Use primer pair (SEQ ID NO:9 and 10) from the mtDNAsublimon amplified target region of disappearance 3.4kb.Forward primer (SEQ ID NO:9) and described 3.4kb sequence end to reconnect site overlapping.

Prostata tissue fixes paraffin-embedded prostata tissue needle puncture biopsy through formalin.

The reagent that this embodiment uses arranges as follows:

In 25 μ l reaction volumes,

Often kind of primer of 250nmol

2 × the reaction mixture of 12.5 μ l,

20ng (10 μ l, 2ng/ μ l) template.

Loop parameter is as follows:

1.95 DEG C 3 minutes

2.95 DEG C 30 seconds

3.62 DEG C 30 seconds

4.72 DEG C 30 seconds

5. read plate

6. repeating step 2-5,44 times

7.72 DEG C 10 minutes

8. melting curve is from 50 DEG C to 100 DEG C, every 1 DEG C of reading, keeps 3 seconds

9. keep at 4 DEG C.

Use primer pair (SEQ ID NO:9 and 10) from the mtDNAsublimon amplified target region of described disappearance 3.4kb.

Hereafter table 16 provide for detect 3.4kb disappearance actual in the mtDNA that obtains from pernicious and benign prostate tissue the summary of test carried out.Use C _tscore value 30.0, likely identifies pernicious and benign tissue significantly.Thus, the increase instruction cancer of the amount of the described 3.4kb molecule existed in sample.

Table 16: for detecting the C of cancer in prostata tissue _tscore value

Describe	C T
		Benign sample 1	33.75
Malignant samples 1	28.79
		Benign sample 2	30.96
Malignant samples 2	28.4
		Benign sample 3	32.19
Malignant samples 3	27.38

Although the present invention is described by some specific embodiments, under the prerequisite of spirit and scope not deviating from the present invention that claims are herein summarized, multiple amendment is obvious to those skilled in the art.

Reference

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Sequence table

<110> Genesis Genomics Inc.

<120>3.4KB mitochondrial DNA deletion is for detecting cancer

<130>102222/00031

<160>10

<170>PatentIn version 3.3

<210>1

<211>3379

<212>DNA

<213> people (Homo sapiens)

<400>1

cctaaaccta ctccaatgct aaaactaatc gtcccaacaa ttatattact accactgaca 60

tgactttcca aaaaacacat aatttgaatc aacacaacca cccacagcct aattattagc 120

atcatccctc tactattttt taaccaaatc aacaacaacc tatttagctg ttccccaacc 180

ttttcctccg accccctaac aacccccctc ctaatactaa ctacctgact cctacccctc 240

acaatcatgg caagccaacg ccacttatcc agtgaaccac tatcacgaaa aaaactctac 300

ctctctatac taatctccct acaaatctcc ttaattataa cattcacagc cacagaacta 360

atcatatttt atatcttctt cgaaaccaca cttatcccca ccttggctat catcacccga 420

tgaggcaacc agccagaacg cctgaacgca ggcacatact tcctattcta caccctagta 480

ggctcccttc ccctactcat cgcactaatt tacactcaca acaccctagg ctcactaaac 540

attctactac tcactctcac tgcccaagaa ctatcaaact cctgagccaa caacttaata 600

tgactagctt acacaatagc ttttatagta aagatacctc tttacggact ccacttatga 660

ctccctaaag cccatgtcga agcccccatc gctgggtcaa tagtacttgc cgcagtactc 720

ttaaaactag gcggctatgg tataatacgc ctcacactca ttctcaaccc cctgacaaaa 780

cacatagcct accccttcct tgtactatcc ctatgaggca taattataac aagctccatc 840

tgcctacgac aaacagacct aaaatcgctc attgcatact cttcaatcag ccacatagcc 900

ctcgtagtaa cagccattct catccaaacc ccctgaagct tcaccggcgc agtcattctc 960

ataatcgccc acgggcttac atcctcatta ctattctgcc tagcaaactc aaactacgaa 1020

cgcactcaca gtcgcatcat aatcctctct caaggacttc aaactctact cccactaata 1080

gctttttgat gacttctagc aagcctcgct aacctcgcct taccccccac tattaaccta 1140

ctgggagaac tctctgtgct agtaaccacg ttctcctgat caaatatcac tctcctactt 1200

acaggactca acatactagt cacagcccta tactccctct acatatttac cacaacacaa 1260

tggggctcac tcacccacca cattaacaac ataaaaccct cattcacacg agaaaacacc 1320

ctcatgttca tacacctatc ccccattctc ctcctatccc tcaaccccga catcattacc 1380

gggttttcct cttgtaaata tagtttaacc aaaacatcag attgtgaatc tgacaacaga 1440

ggcttacgac cccttattta ccgagaaagc tcacaagaac tgctaactca tgcccccatg 1500

tctaacaaca tggctttctc aacttttaaa ggataacagc tatccattgg tcttaggccc 1560

caaaaatttt ggtgcaactc caaataaaag taataaccat gcacactact ataaccaccc 1620

taaccctgac ttccctaatt ccccccatcc ttaccaccct cgttaaccct aacaaaaaaa 1680

actcataccc ccattatgta aaatccattg tcgcatccac ctttattatc agtctcttcc 1740

ccacaacaat attcatgtgc ctagaccaag aagttattat ctcgaactga cactgagcca 1800

caacccaaac aacccagctc tccctaagct tcaaactaga ctacttctcc ataatattca 1860

tccctgtagc attgttcgtt acatggtcca tcatagaatt ctcactgtga tatataaact 1920

cagacccaaa cattaatcag ttcttcaaat atctactcat cttcctaatt accatactaa 1980

tcttagttac cgctaacaac ctattccaac tgttcatcgg ctgagagggc gtaggaatta 2040

tatccttctt gctcatcagt tgatgatacg cccgagcaga tgccaacaca gcagccattc 2100

aagcaatcct atacaaccgt atcggcgata tcggtttcat cctcgcctta gcatgattta 2160

tcctacactc caactcatga gacccacaac aaatagccct tctaaacgct aatccaagcc 2220

tcaccccact actaggcctc ctcctagcag cagcaggcaa atcagcccaa ttaggtctcc 2280

acccctgact cccctcagcc atagaaggcc ccaccccagt ctcagcccta ctccactcaa 2340

gcactatagt tgtagcagga atcttcttac tcatccgctt ccacccccta gcagaaaata 2400

gcccactaat ccaaactcta acactatgct taggcgctat caccactctg ttcgcagcag 2460

tctgcgccct tacacaaaat gacatcaaaa aaatcgtagc cttctccact tcaagtcaac 2520

taggactcat aatagttaca atcggcatca accaaccaca cctagcattc ctgcacatct 2580

gtacccacgc cttcttcaaa gccatactat ttatgtgctc cgggtccatc atccacaacc 2640

ttaacaatga acaagatatt cgaaaaatag gaggactact caaaaccata cctctcactt 2700

caacctccct caccattggc agcctagcat tagcaggaat acctttcctc acaggtttct 2760

actccaaaga ccacatcatc gaaaccgcaa acatatcata cacaaacgcc tgagccctat 2820

ctattactct catcgctacc tccctgacaa gcgcctatag cactcgaata attcttctca 2880

ccctaacagg tcaacctcgc ttccccaccc ttactaacat taacgaaaat aaccccaccc 2940

tactaaaccc cattaaacgc ctggcagccg gaagcctatt cgcaggattt ctcattacta 3000

acaacatttc ccccgcatcc cccttccaaa caacaatccc cctctaccta aaactcacag 3060

ccctcgctgt cactttccta ggacttctaa cagccctaga cctcaactac ctaaccaaca 3120

aacttaaaat aaaatcccca ctatgcacat tttatttctc caacatactc ggattctacc 3180

ctagcatcac acaccgcaca atcccctatc taggccttct tacgagccaa aacctgcccc 3240

tactcctcct agacctaacc tgactagaaa agctattacc taaaacaatt tcacagcacc 3300

aaatctccac ctccatcatc acctcaaccc aaaaaggcat aattaaactt tacttcctct 3360

ctttcttctt cccactcat 3379

<210>2

<211>30

<212>DNA

<213> is artificial

<220>

<223>3.4kb lacks forward and detects primer

<400>2

tagactacgt acatactaac cctactccta 30

<210>3

<211>19

<212>DNA

<213> is artificial

<220>

<223>3.4mtDNA lacks inverse detection primer

<400>3

gaggtaggat tggtgctgt 19

<210>4

<211>21

<212>DNA

<213> is artificial

<220>

<223>mtDNA genome forward primer

<400>4

cgttccagtg agttcaccct c 21

<210>5

<211>23

<212>DNA

<213> is artificial

<220>

<223>mtDNA is because of group reverse primer

<400>5

cactctttac gccggcttctatt 23

<210>6

<211>20

<212>DNA

<213> is artificial

<220>

<223>TNF nuclear gene forward primer

<400>6

cctgccccaa tccctttatt 20

<210>7

<211>21

<212>DNA

<213> is artificial

<220>

<223>TNF nuclear gene reverse primer

<400>7

ggtttcgaag tggtggtctt g 21

<210>8

<211>16569

<212>DNA

<213> people

<220>

<221>misc_feature

<222>(3107)..(3107)

<223>n is a, c, g, or t

<300>

<308>AC_000021

<309>2007-03-07

<313>(1)..(16569)

<400>8

gatcacaggt ctatcaccct attaaccact cacgggagct ctccatgcat ttggtatttt 60

cgtctggggg gtatgcacgc gatagcattg cgagacgctg gagccggagc accctatgtc 120

gcagtatctg tctttgattc ctgcctcatc ctattattta tcgcacctac gttcaatatt 180

acaggcgaac atacttacta aagtgtgtta attaattaat gcttgtagga cataataata 240

acaattgaat gtctgcacag ccactttcca cacagacatc ataacaaaaa atttccacca 300

aaccccccct cccccgcttc tggccacagc acttaaacac atctctgcca aaccccaaaa 360

acaaagaacc ctaacaccag cctaaccaga tttcaaattt tatcttttgg cggtatgcac 420

ttttaacagt caccccccaa ctaacacatt attttcccct cccactccca tactactaat 480

ctcatcaata caacccccgc ccatcctacc cagcacacac acaccgctgc taaccccata 540

ccccgaacca accaaacccc aaagacaccc cccacagttt atgtagctta cctcctcaaa 600

gcaatacact gaaaatgttt agacgggctc acatcacccc ataaacaaat aggtttggtc 660

ctagcctttc tattagctct tagtaagatt acacatgcaa gcatccccgt tccagtgagt 720

tcaccctcta aatcaccacg atcaaaagga acaagcatca agcacgcagc aatgcagctc 780

aaaacgctta gcctagccac acccccacgg gaaacagcag tgattaacct ttagcaataa 840

acgaaagttt aactaagcta tactaacccc agggttggtc aatttcgtgc cagccaccgc 900

ggtcacacga ttaacccaag tcaatagaag ccggcgtaaa gagtgtttta gatcaccccc 960

tccccaataa agctaaaact cacctgagtt gtaaaaaact ccagttgaca caaaatagac 1020

tacgaaagtg gctttaacat atctgaacac acaatagcta agacccaaac tgggattaga 1080

taccccacta tgcttagccc taaacctcaa cagttaaatc aacaaaactg ctcgccagaa 1140

cactacgagc cacagcttaa aactcaaagg acctggcggt gcttcatatc cctctagagg 1200

agcctgttct gtaatcgata aaccccgatc aacctcacca cctcttgctc agcctatata 1260

ccgccatctt cagcaaaccc tgatgaaggc tacaaagtaa gcgcaagtac ccacgtaaag 1320

acgttaggtc aaggtgtagc ccatgaggtg gcaagaaatg ggctacattt tctaccccag 1380

aaaactacga tagcccttat gaaacttaag ggtcgaaggt ggatttagca gtaaactaag 1440

agtagagtgc ttagttgaac agggccctga agcgcgtaca caccgcccgt caccctcctc 1500

aagtatactt caaaggacat ttaactaaaa cccctacgca tttatataga ggagacaagt 1560

cgtaacatgg taagtgtact ggaaagtgca cttggacgaa ccagagtgta gcttaacaca 1620

aagcacccaa cttacactta ggagatttca acttaacttg accgctctga gctaaaccta 1680

gccccaaacc cactccacct tactaccaga caaccttagc caaaccattt acccaaataa 1740

agtataggcg atagaaattg aaacctggcg caatagatat agtaccgcaa gggaaagatg 1800

aaaaattata accaagcata atatagcaag gactaacccc tataccttct gcataatgaa 1860

ttaactagaa ataactttgc aaggagagcc aaagctaaga cccccgaaac cagacgagct 1920

acctaagaac agctaaaaga gcacacccgt ctatgtagca aaatagtggg aagatttata 1980

ggtagaggcg acaaacctac cgagcctggt gatagctggt tgtccaagat agaatcttag 2040

ttcaacttta aatttgccca cagaaccctc taaatcccct tgtaaattta actgttagtc 2100

caaagaggaa cagctctttg gacactagga aaaaaccttg tagagagagt aaaaaattta 2160

acacccatag taggcctaaa agcagccacc aattaagaaa gcgttcaagc tcaacaccca 2220

ctacctaaaa aatcccaaac atataactga actcctcaca cccaattgga ccaatctatc 2280

accctataga agaactaatg ttagtataag taacatgaaa acattctcct ccgcataagc 2340

ctgcgtcaga ttaaaacact gaactgacaa ttaacagccc aatatctaca atcaaccaac 2400

aagtcattat taccctcact gtcaacccaa cacaggcatg ctcataagga aaggttaaaa 2460

aaagtaaaag gaactcggca aatcttaccc cgcctgttta ccaaaaacat cacctctagc 2520

atcaccagta ttagaggcac cgcctgccca gtgacacatg tttaacggcc gcggtaccct 2580

aaccgtgcaa aggtagcata atcacttgtt ccttaaatag ggacctgtat gaatggctcc 2640

acgagggttc agctgtctct tacttttaac cagtgaaatt gacctgcccg tgaagaggcg 2700

ggcataacac agcaagacga gaagacccta tggagcttta atttattaat gcaaacagta 2760

cctaacaaac ccacaggtcc taaactacca aacctgcatt aaaaatttcg gttggggcga 2820

cctcggagca gaacccaacc tccgagcagt acatgctaag acttcaccag tcaaagcgaa 2880

ctactatact caattgatcc aataacttga ccaacggaac aagttaccct agggataaca 2940

gcgcaatcct attctagagt ccatatcaac aatagggttt acgacctcga tgttggatca 3000

ggacatcccg atggtgcagc cgctattaaa ggttcgtttg ttcaacgatt aaagtcctac 3060

gtgatctgag ttcagaccgg agtaatccag gtcggtttct atctacnttc aaattcctcc 3120

ctgtacgaaa ggacaagaga aataaggcct acttcacaaa gcgccttccc ccgtaaatga 3180

tatcatctca acttagtatt atacccacac ccacccaaga acagggtttg ttaagatggc 3240

agagcccggt aatcgcataa aacttaaaac tttacagtca gaggttcaat tcctcttctt 3300

aacaacatac ccatggccaa cctcctactc ctcattgtac ccattctaat cgcaatggca 3360

ttcctaatgc ttaccgaacg aaaaattcta ggctatatac aactacgcaa aggccccaac 3420

gttgtaggcc cctacgggct actacaaccc ttcgctgacg ccataaaact cttcaccaaa 3480

gagcccctaa aacccgccac atctaccatc accctctaca tcaccgcccc gaccttagct 3540

ctcaccatcg ctcttctact atgaaccccc ctccccatac ccaaccccct ggtcaacctc 3600

aacctaggcc tcctatttat tctagccacc tctagcctag ccgtttactc aatcctctga 3660

tcagggtgag catcaaactc aaactacgcc ctgatcggcg cactgcgagc agtagcccaa 3720

acaatctcat atgaagtcac cctagccatc attctactat caacattact aataagtggc 3780

tcctttaacc tctccaccct tatcacaaca caagaacacc tctgattact cctgccatca 3840

tgacccttgg ccataatatg atttatctcc acactagcag agaccaaccg aacccccttc 3900

gaccttgccg aaggggagtc cgaactagtc tcaggcttca acatcgaata cgccgcaggc 3960

cccttcgccc tattcttcat agccgaatac acaaacatta ttataataaa caccctcacc 4020

actacaatct tcctaggaac aacatatgac gcactctccc ctgaactcta cacaacatat 4080

tttgtcacca agaccctact tctaacctcc ctgttcttat gaattcgaac agcatacccc 4140

cgattccgct acgaccaact catacacctc ctatgaaaaa acttcctacc actcacccta 4200

gcattactta tatgatatgt ctccataccc attacaatct ccagcattcc ccctcaaacc 4260

taagaaatat gtctgataaa agagttactt tgatagagta aataatagga gcttaaaccc 4320

ccttatttct aggactatga gaatcgaacc catccctgag aatccaaaat tctccgtgcc 4380

acctatcaca ccccatccta aagtaaggtc agctaaataa gctatcgggc ccataccccg 4440

aaaatgttgg ttataccctt cccgtactaa ttaatcccct ggcccaaccc gtcatctact 4500

ctaccatctt tgcaggcaca ctcatcacag cgctaagctc gcactgattt tttacctgag 4560

taggcctaga aataaacatg ctagctttta ttccagttct aaccaaaaaa ataaaccctc 4620

gttccacaga agctgccatc aagtatttcc tcacgcaagc aaccgcatcc ataatccttc 4680

taatagctat cctcttcaac aatatactct ccggacaatg aaccataacc aatactacca 4740

atcaatactc atcattaata atcataatag ctatagcaat aaaactagga atagccccct 4800

ttcacttctg agtcccagag gttacccaag gcacccctct gacatccggc ctgcttcttc 4860

tcacatgaca aaaactagcc cccatctcaa tcatatacca aatctctccc tcactaaacg 4920

taagccttct cctcactctc tcaatcttat ccatcatagc aggcagttga ggtggattaa 4980

accaaaccca gctacgcaaa atcttagcat actcctcaat tacccacata ggatgaataa 5040

tagcagttct accgtacaac cctaacataa ccattcttaa tttaactatt tatattatcc 5100

taactactac cgcattccta ctactcaact taaactccag caccacgacc ctactactat 5160

ctcgcacctg aaacaagcta acatgactaa cacccttaat tccatccacc ctcctctccc 5220

taggaggcct gcccccgcta accggctttt tgcccaaatg ggccattatc gaagaattca 5280

caaaaaacaa tagcctcatc atccccacca tcatagccac catcaccctc cttaacctct 5340

acttctacct acgcctaatc tactccacct caatcacact actccccata tctaacaacg 5400

taaaaataaa atgacagttt gaacatacaa aacccacccc attcctcccc acactcatcg 5460

cccttaccac gctactccta cctatctccc cttttatact aataatctta tagaaattta 5520

ggttaaatac agaccaagag ccttcaaagc cctcagtaag ttgcaatact taatttctgt 5580

aacagctaag gactgcaaaa ccccactctg catcaactga acgcaaatca gccactttaa 5640

ttaagctaag cccttactag accaatggga cttaaaccca caaacactta gttaacagct 5700

aagcacccta atcaactggc ttcaatctac ttctcccgcc gccgggaaaa aaggcgggag 5760

aagccccggc aggtttgaag ctgcttcttc gaatttgcaa ttcaatatga aaatcacctc 5820

ggagctggta aaaagaggcc taacccctgt ctttagattt acagtccaat gcttcactca 5880

gccattttac ctcaccccca ctgatgttcg ccgaccgttg actattctct acaaaccaca 5940

aagacattgg aacactatac ctattattcg gcgcatgagc tggagtccta ggcacagctc 6000

taagcctcct tattcgagcc gagctgggcc agccaggcaa ccttctaggt aacgaccaca 6060

tctacaacgt tatcgtcaca gcccatgcat ttgtaataat cttcttcata gtaataccca 6120

tcataatcgg aggctttggc aactgactag ttcccctaat aatcggtgcc cccgatatgg 6180

cgtttccccg cataaacaac ataagcttct gactcttacc tccctctctc ctactcctgc 6240

tcgcatctgc tatagtggag gccggagcag gaacaggttg aacagtctac cctcccttag 6300

cagggaacta ctcccaccct ggagcctccg tagacctaac catcttctcc ttacacctag 6360

caggtgtctc ctctatctta ggggccatca atttcatcac aacaattatc aatataaaac 6420

cccctgccat aacccaatac caaacgcccc tcttcgtctg atccgtccta atcacagcag 6480

tcctacttct cctatctctc ccagtcctag ctgctggcat cactatacta ctaacagacc 6540

gcaacctcaa caccaccttc ttcgaccccg ccggaggagg agaccccatt ctataccaac 6600

acctattctg atttttcggt caccctgaag tttatattct tatcctacca ggcttcggaa 6660

taatctccca tattgtaact tactactccg gaaaaaaaga accatttgga tacataggta 6720

tggtctgagc tatgatatca attggcttcc tagggtttat cgtgtgagca caccatatat 6780

ttacagtagg aatagacgta gacacacgag catatttcac ctccgctacc ataatcatcg 6840

ctatccccac cggcgtcaaa gtatttagct gactcgccac actccacgga agcaatatga 6900

aatgatctgc tgcagtgctc tgagccctag gattcatctt tcttttcacc gtaggtggcc 6960

tgactggcat tgtattagca aactcatcac tagacatcgt actacacgac acgtactacg 7020

ttgtagccca cttccactat gtcctatcaa taggagctgt atttgccatc ataggaggct 7080

tcattcactg atttccccta ttctcaggct acaccctaga ccaaacctac gccaaaatcc 7140

atttcactat catattcatc ggcgtaaatc taactttctt cccacaacac tttctcggcc 7200

tatccggaat gccccgacgt tactcggact accccgatgc atacaccaca tgaaacatcc 7260

tatcatctgt aggctcattc atttctctaa cagcagtaat attaataatt ttcatgattt 7320

gagaagcctt cgcttcgaag cgaaaagtcc taatagtaga agaaccctcc ataaacctgg 7380

agtgactata tggatgcccc ccaccctacc acacattcga agaacccgta tacataaaat 7440

ctagacaaaa aaggaaggaa tcgaaccccc caaagctggt ttcaagccaa ccccatggcc 7500

tccatgactt tttcaaaaag gtattagaaa aaccatttca taactttgtc aaagttaaat 7560

tataggctaa atcctatata tcttaatggc acatgcagcg caagtaggtc tacaagacgc 7620

tacttcccct atcatagaag agcttatcac ctttcatgat cacgccctca taatcatttt 7680

ccttatctgc ttcctagtcc tgtatgccct tttcctaaca ctcacaacaa aactaactaa 7740

tactaacatc tcagacgctc aggaaataga aaccgtctga actatcctgc ccgccatcat 7800

cctagtcctc atcgccctcc catccctacg catcctttac ataacagacg aggtcaacga 7860

tccctccctt accatcaaat caattggcca ccaatggtac tgaacctacg agtacaccga 7920

ctacggcgga ctaatcttca actcctacat acttccccca ttattcctag aaccaggcga 7980

cctgcgactc cttgacgttg acaatcgagt agtactcccg attgaagccc ccattcgtat 8040

aataattaca tcacaagacg tcttgcactc atgagctgtc cccacattag gcttaaaaac 8100

agatgcaatt cccggacgtc taaaccaaac cactttcacc gctacacgac cgggggtata 8160

ctacggtcaa tgctctgaaa tctgtggagc aaaccacagt ttcatgccca tcgtcctaga 8220

attaattccc ctaaaaatct ttgaaatagg gcccgtattt accctatagc accccctcta 8280

ccccctctag agcccactgt aaagctaact tagcattaac cttttaagtt aaagattaag 8340

agaaccaaca cctctttaca gtgaaatgcc ccaactaaat actaccgtat ggcccaccat 8400

aattaccccc atactcctta cactattcct catcacccaa ctaaaaatat taaacacaaa 8460

ctaccaccta cctccctcac caaagcccat aaaaataaaa aattataaca aaccctgaga 8520

accaaaatga acgaaaatct gttcgcttca ttcattgccc ccacaatcct aggcctaccc 8580

gccgcagtac tgatcattct atttccccct ctattgatcc ccacctccaa atatctcatc 8640

aacaaccgac taatcaccac ccaacaatga ctaatcaaac taacctcaaa acaaatgata 8700

accatacaca acactaaagg acgaacctga tctcttatac tagtatcctt aatcattttt 8760

attgccacaa ctaacctcct cggactcctg cctcactcat ttacaccaac cacccaacta 8820

tctataaacc tagccatggc catcccctta tgagcgggca cagtgattat aggctttcgc 8880

tctaagatta aaaatgccct agcccacttc ttaccacaag gcacacctac accccttatc 8940

cccatactag ttattatcga aaccatcagc ctactcattc aaccaatagc cctggccgta 9000

cgcctaaccg ctaacattac tgcaggccac ctactcatgc acctaattgg aagcgccacc 9060

ctagcaatat caaccattaa ccttccctct acacttatca tcttcacaat tctaattcta 9120

ctgactatcc tagaaatcgc tgtcgcctta atccaagcct acgttttcac acttctagta 9180

agcctctacc tgcacgacaa cacataatga cccaccaatc acatgcctat catatagtaa 9240

aacccagccc atgaccccta acaggggccc tctcagccct cctaatgacc tccggcctag 9300

ccatgtgatt tcacttccac tccataacgc tcctcatact aggcctacta accaacacac 9360

taaccatata ccaatgatgg cgcgatgtaa cacgagaaag cacataccaa ggccaccaca 9420

caccacctgt ccaaaaaggc cttcgatacg ggataatcct atttattacc tcagaagttt 9480

ttttcttcgc aggatttttc tgagcctttt accactccag cctagcccct accccccaat 9540

taggagggca ctggccccca acaggcatca ccccgctaaa tcccctagaa gtcccactcc 9600

taaacacatc cgtattactc gcatcaggag tatcaatcac ctgagctcac catagtctaa 9660

tagaaaacaa ccgaaaccaa ataattcaag cactgcttat tacaatttta ctgggtctct 9720

attttaccct cctacaagcc tcagagtact tcgagtctcc cttcaccatt tccgacggca 9780

tctacggctc aacatttttt gtagccacag gcttccacgg acttcacgtc attattggct 9840

caactttcct cactatctgc ttcatccgcc aactaatatt tcactttaca tccaaacatc 9900

actttggctt cgaagccgcc gcctgatact ggcattttgt agatgtggtt tgactatttc 9960

tgtatgtctc catctattga tgagggtctt actcttttag tataaatagt accgttaact 10020

tccaattaac tagttttgac aacattcaaa aaagagtaat aaacttcgcc ttaattttaa 10080

taatcaacac cctcctagcc ttactactaa taattattac attttgacta ccacaactca 10140

acggctacat agaaaaatcc accccttacg agtgcggctt cgaccctata tcccccgccc 10200

gcgtcccttt ctccataaaa ttcttcttag tagctattac cttcttatta tttgatctag 10260

aaattgccct ccttttaccc ctaccatgag ccctacaaac aactaacctg ccactaatag 10320

ttatgtcatc cctcttatta atcatcatcc tagccctaag tctggcctat gagtgactac 10380

aaaaaggatt agactgaacc gaattggtat atagtttaaa caaaacgaat gatttcgact 10440

cattaaatta tgataatcat atttaccaaa tgcccctcat ttacataaat attatactag 10500

catttaccat ctcacttcta ggaatactag tatatcgctc acacctcata tcctccctac 10560

tatgcctaga aggaataata ctatcgctgt tcattatagc tactctcata accctcaaca 10620

cccactccct cttagccaat attgtgccta ttgccatact agtctttgcc gcctgcgaag 10680

cagcggtggg cctagcccta ctagtctcaa tctccaacac atatggccta gactacgtac 10740

ataacctaaa cctactccaa tgctaaaact aatcgtccca acaattatat tactaccact 10800

gacatgactt tccaaaaaac acataatttg aatcaacaca accacccaca gcctaattat 10860

tagcatcatc cctctactat tttttaacca aatcaacaac aacctattta gctgttcccc 10920

aaccttttcc tccgaccccc taacaacccc cctcctaata ctaactacct gactcctacc 10980

cctcacaatc atggcaagcc aacgccactt atccagtgaa ccactatcac gaaaaaaact 11040

ctacctctct atactaatct ccctacaaat ctccttaatt ataacattca cagccacaga 11100

actaatcata ttttatatct tcttcgaaac cacacttatc cccaccttgg ctatcatcac 11160

ccgatgaggc aaccagccag aacgcctgaa cgcaggcaca tacttcctat tctacaccct 11220

agtaggctcc cttcccctac tcatcgcact aatttacact cacaacaccc taggctcact 11280

aaacattcta ctactcactc tcactgccca agaactatca aactcctgag ccaacaactt 11340

aatatgacta gcttacacaa tagcttttat agtaaagata cctctttacg gactccactt 11400

atgactccct aaagcccatg tcgaagcccc catcgctggg tcaatagtac ttgccgcagt 11460

actcttaaaa ctaggcggct atggtataat acgcctcaca ctcattctca accccctgac 11520

aaaacacata gcctacccct tccttgtact atccctatga ggcataatta taacaagctc 11580

catctgccta cgacaaacag acctaaaatc gctcattgca tactcttcaa tcagccacat 11640

agccctcgta gtaacagcca ttctcatcca aaccccctga agcttcaccg gcgcagtcat 11700

tctcataatc gcccacgggc ttacatcctc attactattc tgcctagcaa actcaaacta 11760

cgaacgcact cacagtcgca tcataatcct ctctcaagga cttcaaactc tactcccact 11820

aatagctttt tgatgacttc tagcaagcct cgctaacctc gccttacccc ccactattaa 11880

cctactggga gaactctctg tgctagtaac cacgttctcc tgatcaaata tcactctcct 11940

acttacagga ctcaacatac tagtcacagc cctatactcc ctctacatat ttaccacaac 12000

acaatggggc tcactcaccc accacattaa caacataaaa ccctcattca cacgagaaaa 12060

caccctcatg ttcatacacc tatcccccat tctcctccta tccctcaacc ccgacatcat 12120

taccgggttt tcctcttgta aatatagttt aaccaaaaca tcagattgtg aatctgacaa 12180

cagaggctta cgacccctta tttaccgaga aagctcacaa gaactgctaa ctcatgcccc 12240

catgtctaac aacatggctt tctcaacttt taaaggataa cagctatcca ttggtcttag 12300

gccccaaaaa ttttggtgca actccaaata aaagtaataa ccatgcacac tactataacc 12360

accctaaccc tgacttccct aattcccccc atccttacca ccctcgttaa ccctaacaaa 12420

aaaaactcat acccccatta tgtaaaatcc attgtcgcat ccacctttat tatcagtctc 12480

ttccccacaa caatattcat gtgcctagac caagaagtta ttatctcgaa ctgacactga 12540

gccacaaccc aaacaaccca gctctcccta agcttcaaac tagactactt ctccataata 12600

ttcatccctg tagcattgtt cgttacatgg tccatcatag aattctcact gtgatatata 12660

aactcagacc caaacattaa tcagttcttc aaatatctac tcatcttcct aattaccata 12720

ctaatcttag ttaccgctaa caacctattc caactgttca tcggctgaga gggcgtagga 12780

attatatcct tcttgctcat cagttgatga tacgcccgag cagatgccaa cacagcagcc 12840

attcaagcaa tcctatacaa ccgtatcggc gatatcggtt tcatcctcgc cttagcatga 12900

tttatcctac actccaactc atgagaccca caacaaatag cccttctaaa cgctaatcca 12960

agcctcaccc cactactagg cctcctccta gcagcagcag gcaaatcagc ccaattaggt 13020

ctccacccct gactcccctc agccatagaa ggccccaccc cagtctcagc cctactccac 13080

tcaagcacta tagttgtagc aggaatcttc ttactcatcc gcttccaccc cctagcagaa 13140

aatagcccac taatccaaac tctaacacta tgcttaggcg ctatcaccac tctgttcgca 13200

gcagtctgcg cccttacaca aaatgacatc aaaaaaatcg tagccttctc cacttcaagt 13260

caactaggac tcataatagt tacaatcggc atcaaccaac cacacctagc attcctgcac 13320

atctgtaccc acgccttctt caaagccata ctatttatgt gctccgggtc catcatccac 13380

aaccttaaca atgaacaaga tattcgaaaa ataggaggac tactcaaaac catacctctc 13440

acttcaacct ccctcaccat tggcagccta gcattagcag gaataccttt cctcacaggt 13500

ttctactcca aagaccacat catcgaaacc gcaaacatat catacacaaa cgcctgagcc 13560

ctatctatta ctctcatcgc tacctccctg acaagcgcct atagcactcg aataattctt 13620

ctcaccctaa caggtcaacc tcgcttcccc acccttacta acattaacga aaataacccc 13680

accctactaa accccattaa acgcctggca gccggaagcc tattcgcagg atttctcatt 13740

actaacaaca tttcccccgc atcccccttc caaacaacaa tccccctcta cctaaaactc 13800

acagccctcg ctgtcacttt cctaggactt ctaacagccc tagacctcaa ctacctaacc 13860

aacaaactta aaataaaatc cccactatgc acattttatt tctccaacat actcggattc 13920

taccctagca tcacacaccg cacaatcccc tatctaggcc ttcttacgag ccaaaacctg 13980

cccctactcc tcctagacct aacctgacta gaaaagctat tacctaaaac aatttcacag 14040

caccaaatct ccacctccat catcacctca acccaaaaag gcataattaa actttacttc 14100

ctctctttct tcttcccact catcctaacc ctactcctaa tcacataacc tattcccccg 14160

agcaatctca attacaatat atacaccaac aaacaatgtt caaccagtaa ctactactaa 14220

tcaacgccca taatcataca aagcccccgc accaatagga tcctcccgaa tcaaccctga 14280

cccctctcct tcataaatta ttcagcttcc tacactatta aagtttacca caaccaccac 14340

cccatcatac tctttcaccc acagcaccaa tcctacctcc atcgctaacc ccactaaaac 14400

actcaccaag acctcaaccc ctgaccccca tgcctcagga tactcctcaa tagccatcgc 14460

tgtagtatat ccaaagacaa ccatcattcc ccctaaataa attaaaaaaa ctattaaacc 14520

catataacct cccccaaaat tcagaataat aacacacccg accacaccgc taacaatcaa 14580

tactaaaccc ccataaatag gagaaggctt agaagaaaac cccacaaacc ccattactaa 14640

acccacactc aacagaaaca aagcatacat cattattctc gcacggacta caaccacgac 14700

caatgatatg aaaaaccatc gttgtatttc aactacaaga acaccaatga ccccaatacg 14760

caaaactaac cccctaataa aattaattaa ccactcattc atcgacctcc ccaccccatc 14820

caacatctcc gcatgatgaa acttcggctc actccttggc gcctgcctga tcctccaaat 14880

caccacagga ctattcctag ccatgcacta ctcaccagac gcctcaaccg ccttttcatc 14940

aatcgcccac atcactcgag acgtaaatta tggctgaatc atccgctacc ttcacgccaa 15000

tggcgcctca atattcttta tctgcctctt cctacacatc gggcgaggcc tatattacgg 15060

atcatttctc tactcagaaa cctgaaacat cggcattatc ctcctgcttg caactatagc 15120

aacagccttc ataggctatg tcctcccgtg aggccaaata tcattctgag gggccacagt 15180

aattacaaac ttactatccg ccatcccata cattgggaca gacctagttc aatgaatctg 15240

aggaggctac tcagtagaca gtcccaccct cacacgattc tttacctttc acttcatctt 15300

gcccttcatt attgcagccc tagcaacact ccacctccta ttcttgcacg aaacgggatc 15360

aaacaacccc ctaggaatca cctcccattc cgataaaatc accttccacc cttactacac 15420

aatcaaagac gccctcggct tacttctctt ccttctctcc ttaatgacat taacactatt 15480

ctcaccagac ctcctaggcg acccagacaa ttatacccta gccaacccct taaacacccc 15540

tccccacatc aagcccgaat gatatttcct attcgcctac acaattctcc gatccgtccc 15600

taacaaacta ggaggcgtcc ttgccctatt actatccatc ctcatcctag caataatccc 15660

catcctccat atatccaaac aacaaagcat aatatttcgc ccactaagcc aatcacttta 15720

ttgactccta gccgcagacc tcctcattct aacctgaatc ggaggacaac cagtaagcta 15780

cccttttacc atcattggac aagtagcatc cgtactatac ttcacaacaa tcctaatcct 15840

aataccaact atctccctaa ttgaaaacaa aatactcaaa tgggcctgtc cttgtagtat 15900

aaactaatac accagtcttg taaaccggag atgaaaacct ttttccaagg acaaatcaga 15960

gaaaaagtct ttaactccac cattagcacc caaagctaag attctaattt aaactattct 16020

ctgttctttc atggggaagc agatttgggt accacccaag tattgactca cccatcaaca 16080

accgctatgt atttcgtaca ttactgccag ccaccatgaa tattgtacgg taccataaat 16140

acttgaccac ctgtagtaca taaaaaccca atccacatca aaaccccctc cccatgctta 16200

caagcaagta cagcaatcaa ccctcaacta tcacacatca actgcaactc caaagccacc 16260

cctcacccac taggatacca acaaacctac ccacccttaa cagtacatag tacataaagc 16320

catttaccgt acatagcaca ttacagtcaa atcccttctc gtccccatgg atgacccccc 16380

tcagataggg gtcccttgac caccatcctc cgtgaaatca atatcccgca caagagtgct 16440

actctcctcg ctccgggccc ataacacttg ggggtagcta aagtgaactg tatccgacat 16500

ctggttccta cttcagggtc ataaagccta aatagcccac acgttcccct taaataagac 16560

atcacgatg 16569

<210>9

<211>22

<212>DNA

<213> is artificial

<220>

<223> is for detecting the forward primer of 3.4kb mtDNA deletion sequence

<400>9

cccactcatc acctaaacctac 22

<210>10

<211>20

<212>DNA

<213> is artificial

<220>

<223> is for the reverse primer of 3.4kb deletion sequence

<400>10

ggtaggagtc aggtagttag 20

Claims (31)

1. the purposes in the test kit of primer pair mammary cancer in for the preparation of detected object, wherein said primer pair is used for increasing Mitochondrial DNA and mtDNA target region in the biological sample obtained from described object, and described target region is the mtDNA region in the montage region had after having lacked mtDNA genome 10744 ~ 14124 Nucleotide.

2. the purposes of claim 1, each member of wherein said primer pair corresponds respectively to SEQ ID NO:2 and SEQ ID NO:3.

3. the purposes of claim 1, wherein comprises the amount in quantitative described sample with the mtDNA of described target region to the detection of mammary cancer, and the amount and the known reference point of at least one in described sample with the mtDNA of described target region is compared.

4. the purposes of claim 3, the known reference point of wherein said at least one is the amount in region of hitting with reference to sample from the mtDNA of known non-cancer tissue or body fluid.

5. the purposes of claim 3, the known reference point of wherein said at least one is the amount in region of hitting with reference to sample from the mtDNA of known cancerous tissue or body fluid.

6. the purposes of claim 3, wherein said quantification steps uses PCR in real time to carry out.

7. the purposes of claim 3, the wherein amount quantitatively comprising first amplified target region and target region quantitative increased subsequently of target region.

8. the purposes of claim 6, wherein said reference point is cycle threshold.

9. the purposes of claim 4, wherein in biological sample, the amount of target region improves and indicates mammary cancer compared with described reference point.

10. the purposes of claim 5, the wherein similar instruction mammary cancer of the level of target region in biological sample compared with described reference point.

The purposes of 11. claims 1, wherein amplimer to one of with the montage region overlapping being connected described disappearance opposite two ends region.

Purposes in the test kit of 12. primer pairs mammary cancer in for the preparation of detected object, wherein said primer pair is used for increasing Mitochondrial DNA and mtDNA target sequence in the biological sample obtained from described object, described target sequence is the absent region between mtDNA genome 10744 ~ 14124 Nucleotide as shown in SEQ ID NO:1, and wherein said target sequence comprises and reconnects site.

The purposes of 13. claims 12, wherein said target sequence corresponds to SEQ ID NO:1.

The purposes of 14. claims 12, each member of wherein said primer pair corresponds respectively to SEQ ID NO:9 and SEQ ID NO:10.

15. the purposes of claim 1 or 12, it also comprises reagent and explanation.

The purposes of 16. claims 1 or 12, it also comprises the device from described object extraction biological sample.

The purposes of 17. claims 12, wherein comprises the amount in quantitative described sample with the mtDNA of described target sequence to the detection of mammary cancer, and the amount and the known reference point of at least one in described sample with the mtDNA of described target sequence is compared.

The purposes of 18. claims 17, the known reference point of wherein said at least one is the amount of sequence of hitting with reference to sample from the mtDNA of known non-cancer tissue or body fluid.

The purposes of 19. claims 17, the known reference point of wherein said at least one is the amount of sequence of hitting with reference to sample from the mtDNA of known cancerous tissue or body fluid.

The purposes of 20. claims 17, wherein said quantification steps uses PCR in real time to carry out.

The purposes of 21. claims 17, the wherein amount quantitatively comprising first amplified target sequence and target region quantitative increased subsequently of target sequence.

The purposes of 22. claims 20, wherein said reference point is cycle threshold.

The purposes of 23. claims 18, wherein in biological sample, the amount of target sequence improves and indicates mammary cancer compared with described reference point.

The purposes of 24. claims 19, the wherein similar instruction mammary cancer of the level of target sequence in biological sample compared with described reference point.

The purposes of 25. claims 12, wherein amplimer to one of with connect opposite two ends region, described disappearance district to reconnect site overlapping.

26. with the purposes in the reagent of at least one primer of the target complement sequence of biological sample Mitochondria DNA and mtDNA mammary cancer in for the preparation of detected object, described target sequence be the mtDNA disappearance of the cyclisation again comprising mtDNA genome 10744 ~ 14124 Nucleotide reconnect site.

The purposes of 27. claims 26, at least one primer wherein said is for increasing described mtDNA target sequence and carrying out quantitatively increased mtDNA.

28. the purposes of claim 26, wherein said mtDNA disappearance has the nucleotide sequence shown in SEQ ID NO:1.

The purposes of 29. claims 26, at least one primer wherein said has the nucleotide sequence shown in SEQ ID NO:9 and SEQ ID NO:10.

The purposes of 30. claims 27, its also comprise by described quantitatively and the known reference point of at least one compare.

The purposes of 31. claims 27, wherein said quantification steps uses PCR in real time to carry out.